The Role Of Transcription Factor Twist I In PPARγ-Mediated Adipocytes Insulin Resistance

Objective:Insulin resistance(IR)is closely related with the occurrence of diabetes mellitus,hypertension,obesity and other metabolic diseases according to edemiological studies,which is their "common soil".To explore the expression levels of transcriptional factor Twist 1 in adipocytes insulin resistance,the effects on insulin sensitivity and the regulation relationship with peroxisome proliferator activated receptor gamma in order to get a understanding of the function of transcription factor Twist 1 in PPAR gamma mediated adipocytes insulin resistance so that it can provide the corresponding target for prevention and treatment of insulin resistance related diseases.Methods:1.The collection of clinical specimens:The patients undergoing elective surgery during 2016-2017 were from Qianfo Hill Hospital,Shandong Province.They were divided into control groups and obese-related IR groups according to body mass index(BMI)and homeostasis model assessment-insulin resistance(HOMA-IR).Then collecting their subcutaneous adipose tissues in surgery and then extracting RNA of tissues to analyze transcriptional levels of transcription factor TWIST 1 and PPARγ by way of polymerase chain reaction and protein expression levels of both through Western blot.2.The establishment and identification of insulin resistance model:3T3-L1 preadipocytes were cultured in vitro and induced differentiation into adipocytes and then lipid droplets were stained with oil red O.IR model was established with the high glucose/insulin method and identified through glucose consumption,glucose uptake and glucose transporter 4(GLUT 4)immunofluorescence.In addition,it was necessary to extract nuclear protein and detect the expression of Twist 1 and PPARy in IR model.3.The effects of Twist 1 on insulin sensitivity:Lentivirus was used to transfect 3T3-L1 adipocytes.Comparing with adipocytes which were infected negative virus particles NC-LV3 and NC-LV5 respectively to explore the influences of interference and overexpression of Twist 1 on insulin sensitivity.4.The impacts of Twist 1 expression on PPARy in adipocytes:Lentiviras was used to transfect 3T3-L1 adipocytes.Then exploring the influences of interference and overexpression of Twist 1 on PPARy in adipocytes through comparing with adipocytes which were infected negative virus particles NC-LV3 and NC-LV5 respectively.5.The influences of PPARγ on Twist 1 in adipocytes:PPARy agonist pioglitazone(0.1μmmol/L)and inhibitor T0070907(100μmmol/L)were worked on induced adipocytes.After that,detecting the changes of Twist 1 by Western blot.Results:1.Taken the transcriptional levels of TWIST 1 and PPARγ in control groups as 100%,the ones in obesity-IR groups were significantly decreased by 41%and 28%respectively and there was significance(P<0.05).2.The expression levels of TWIST 1 and PPARy were down-regulated by 26%and 20%respectively and the degree of down-regulation had statistical difference(P<0.05)according to semi-quantitative results in subcutaneous adipose tissue of obese-related IR groups on the basis of regarding the levels of control groups as 100%.3.Oil red O staining showed that a number of orange-red lipid droplets were accumulated in plasma after 3T3-L1 preadipocytes were induced differentiation into adipocytes but preadipocytes were uncolored.Taken the non-stimulated condition as 100%,glucose consumption and intake of stimulated groups were 2-fold and 3.12-fold higher than that in non-stimulated ones in control groups,there was significant difference(P<0.05).After IR model was established by the high glucose/insulin method,glucose consumption and intake in stimulated groups were only 1.1 times and 1.3 times higher than that in non-stimulated ones and had no significant change between them(P>0.05).However,glucose consumption,uptake were reduced by 46%and 59%in IR model compared with control groups under low concentrations of insulin and there was significant difference(P<0.05).Moreover,the fluorescence intensity of GLUT 4 on the cell membrane was weaker in IR than that of the control groups under low concentrations of insulin.The protein expression levels of Twist 1 and PPARγ in IR model were significantly 2.2-fold and 1.6-fold higher than those of the control groups,there was statistical significance(P<0.05).4.The corresponding protein levels of Twist 1 were significantly reduced by 50%with the interference and semi-quantitative results showed reduced levels had significant difference(P<0.05)under the condition that cells were infected with negative virus particles NC-LV3 was regarded as control.Comparing Twist 1-/IR groups with NC-LV3/IR groups under the action of insulin,glucose consumption and glucose uptake were increased 1.5 times and 2 times higher,and then there were significant differences(P<0.05).Immunocytochemical staining also showed that the number of GLUT 4 on cell membrane was much more in Twist 1-/IR groups than that in NC-LV3/IR.5.Taken cells infected with NC-LV5 virus particles as negative control,the levels of Twist 1 protein with overexpression was significantly up-regulated by 40%and there was significant difference through semi-quantitative results(P<0.05).But compared with NC-LV5/IR cells,there was no significant difference in glucose consumption,uptake and GLUT 4 fluorescence of Twist+/IR cells,and there was no statistical significance(P>0.05).6.Regard cells infected with NC-LV3 and NC-LV5 negative virus particles as control,the expression of PPARγ in Twist 1-groups was 36%lower than that of control groups and the down-regulated degree had significant difference(P<0.05).Besides,the expression of PPARy in Twist 1+ group was increased by 25%and had significant difference(P<0.05)in the semi-quantitative analysis.7.Compared with the control,the protein expression of Twist 1 in the PPARy agonist pioglitazone was up-regulated by 63%and semi-quantitative results showed that there was significant difference(P<0.05).Twist 1 expression levels was down-regulated by 77%in inhibitor T0070907 groups and the reduced degree had statistical difference(P<0.05).Conclusion:The transcriptional factor Twist 1 was highly expressed in insulin resistance,and the interference of Twist 1 expression could significantly improve the insulin resistance of adipose adipocytes.There was a certain positive regulatory relationship between Twist 1 and PPARy.